Remedy for cancer

ABSTRACT

The present invention is directed to a therapeutic drug for cancer, containing, as an active ingredient, dendritic cells stimulated with an antigen derived from vascular endothelial cells which have been cultured in a medium containing a cancer cell culture supernatant.

TECHNICAL FIELD

The present invention relates to cancer immunotherapy targeting vascularendothelial cells of tumor tissue.

BACKGROUND ART

A variety of approaches have been followed for treatment of cancers,including surgical therapy, chemotherapy, and radiotherapy. Each ofthese approaches has its own merits, and therefore, after consideringtheir characteristics, practitioners select a method that is consideredto be the most effective against the cancer to be treated. Surgicaltherapy is an effective method for cancerous tissues having a certainsize or more. However, surgical invasion causes patients to suffer greatpain and discomfort, and in addition, has some limitations. For example,there are cases where the lesion site is in a location difficult toaccess for removal, as in the case of brain tumors. Also, small cancershaving a size of several millimeters are difficult to locate.Chemotherapy is primarily performed through injection or oraladministration, which are less burdensome to patients. Moreover, sinceanticancer drugs are delivered via blood flow, small tumors can betreated. However, anticancer drugs are essentially toxic to cells,producing extremely severe side effects. Restrictions on dosage andadministration period of such drugs often result in unsatisfactoryresults. In radiotherapy, pin-pointed irradiation of the target sitewith radioactive rays is sometimes difficult, and in addition,surrounding healthy cells are also affected by such irradiation.

As described above, each of these therapeutic methods has its strongpoints and weak points. Therefore, in many cases, these therapies arenot performed solely but are combined appropriately for the treatment ofcancer. Yet, cancer remains as the leading cause of death in Japan, andtherefore, establishment of a novel cancer therapeutic method is apressing need.

In addition to the above three conventional cancer therapies, there is afourth approach; namely, cancer immunotherapy. This approach aims toeliminate cancer through the immune system, which is a natural systemintrinsically possessed by the living subjects for expelling aliensubstances. In early cancer immunotherapy, a cancer vaccine was preparedfrom disrupted cancer cells or a cancer-specific antigen, along with anadjuvant or a similar material, and the vaccine was administered to asubject with an aim to activate cytotoxic T lymphocytes (CTLs). Althoughthis was expected to achieve successful elimination of cancerous tissue,in reality, virtually no favorable results have been obtained therefrom,as cancer cells per se have been derived from the patient's own cellsand thus have very low antigenicity.

In recent years, roles of dendritic cells have been elucidated, and haveattracted attention as providing a breakthrough for novel cancerimmunotherapy. Among different types of biological cells, dendriticcells exhibit the strongest antigen-presenting ability in a livingorganism. In such a dendritic-cell-based immunotherapy, dendritic cellsare pulsed with crushed cancer cell fragments or a cancer specificantigen in an ex vivo system, and the resultant dendritic cells arereturned to a living subject, where the dendritic cells activate CTLsand the immunization system of the subject destroys cancerous tissue.Such cancer immunotherapy making use of dendritic cells not only hasbeen performed on an animal experiment basis, but successful resultshave been reported in human clinical settings. Thus, thedendritic-cell-based immunotherapy has become of interest for itspotential and effectiveness as a promising novel cancer therapy (J.Immunol., 156, p. 2918 (1996), J. Exp. Med., 183, p. 7 (1996), Blood,84, p. 3054 (1994), Immunol., 95, p. 141 (1998), J. Exp. Med., 185, p.1101 (1997), J. Exp. Med., 187, p. 1019 (1998), Blood, 93, p. 780(1999), Science, 283, p. 1183 (1999), J. Exp. Med, 185, p. 1101 (1997)).

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a novel cancerimmunotherapy making use of the functions of dendritic cells.

The present inventors, realizing that tumor tissue grows at a highercell proliferation rate than does healthy tissue and thus inevitablyrequires neovascularization to secure the pathway for enabling thesupply of nutrients and discharge of wastes therethrough, have foundthat dendritic cells which are useful in the production of a drug forcancer immunotherapy targeting vascular endothelial cells of tumortissue can be obtained through incubation of vascular endothelial cellsin a medium containing a supernatant of the cancer cell culture,followed by stimulation of the dendritic cells with the thus-createdvascular-endothelial-cell-derived antigen. The present invention hasbeen achieved on a basis of this finding.

Accordingly, the present invention provides a therapeutic drug forcancer containing, as an active ingredient, dendritic cells stimulatedwith an antigen derived from vascular endothelial cells which have beencultured in a medium containing a cancer cell culture supernatant.

The present invention also provides use, in the production of atherapeutic drug for cancer, of dendritic cells stimulated with anantigen derived from vascular endothelial cells which have been culturedin a medium containing a cancer cell culture supernatant.

The present invention also provides a therapeutic method for cancer,characterized by administering dendritic cells stimulated with anantigen derived from vascular endothelial cells which have been culturedin a medium containing a cancer cell culture supernatant.

The cancer remedy according to the present invention belongs to a cancerimmunotherapy targeting not tumor cells themselves but vascularendothelial cells of tumor tissue. Therefore, the present invention hasthe following advantages over conventional types of cancerimmunotherapy.

(1) The ratio between the two types of cells forming a mass of tumortissue is cancer cells:vascular endothelial cells=100 to 1000:1, whichmeans that a single vascular endothelial cell supports 100 to 1000cancer cells. Therefore, one death incidence of a vascular endothelialcell is expected to lead to the death of 100 to 1000 cancer cells. Itfollows that if vascular endothelial cells are targeted, a 100- to1000-fold efficiency would be obtained as compared with the case wherecancer cells themselves are targeted.

(2) According to the conventional cancer immunotherapy, in the case oftreatment of primary carcinoma, therapeutic effect can be expected onlywhen immunization is carried out by use of cells specific to the cancerspecies of interest; i.e., liver cancer cells for the treatment of livercancer, and lung cancer cells for the treatment of lung cancer. However,when vascular endothelial cells are targeted, the above problem can beavoided; irrespective of whether the cancer to be treated is, forexample, liver cancer or lung cancer, antigens are derived from vascularendothelial cells, and so long as such vascular endothelial cells havebeen under influence of cancer cells, the cells have commoncharacteristics irrespective of cancer species and express commonantigen, proving validity of a single remedy for any cancer species.

(3) In the case of an adult, other than tumor tissues, the only sitewhere active neovascularization takes place in the living body is thesite of a wound which is in the healing process, and therefore, whencancerous vascular endothelial cells are targeted, very high tumorselectivity is expected, with reduced side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the therapeutic effect, on mouse lung cancer,of dendritic cells stimulated with an antigen derived from vascularendothelial cells which have been cultured in a medium containing a B16cancer cell culture supernatant, wherein the effect is shown on thebasis of lung weight. In this FIGURE, the “None” group indicates thecase where B16 alone was administered; the “None pulsed DC” groupindicates the case where B16 and dendritic cells which had not beenpulsed were administered; the “BAEC pulsed DC” group indicates the casewhere BAEC-pulsed dendritic cells were administered; and the “B16CM-BAEC pulsed DC” indicates the case where B16 and dendritic cellswhich had been pulsed with BAEC cultured in a B16 culture supernatantwere administered.

BEST MODE FOR CARRYING OUT THE INVENTION

The dendritic cells serving as an active ingredient of the cancertherapeutic agent of the present invention take vascular endothelialcells of tumor tissue as their targets. For this reason, the vascularendothelial cells to be used for stimulating dendritic cells are thosewhich have been cultured in a medium containing a cancer cell culturesupernatant.

The cancer cell is preferably a human cancer cell. However, noparticular limitation is imposed on the species of the cancer cell.Examples of the cancer cell includes cells of solid cancer, such asgastric cancer, liver cancer, colon cancer, lung cancer, skin cancer,bladder cancer, uterine cervical cancer, ovarian cancer, breast cancer,prostatic cancer, and brain tumor. No particular limitation is imposedon the culture conditions under which the cancer cell culture isperformed. For example, culturing is preferably performed in a mediumsuch as RPMI 1640, MEM, DMEM, Ham's F12, or M199, at 37° C., in anatmosphere of 5% CO₂ and 95% air, under saturated water vaporconditions. The medium may be supplemented with an antibiotic, sodiumhydrogencarbonate, a non-essential amino acid, or another additive. Thesupernatant of a cancer cell culture may be collected throughcentrifugation performed, for example, at 500 to 1,500 rpm for 2 to 10minutes, and filtration.

No particular limitation is imposed on the species of the source ofvascular endothelial cells. For example, vascular endothelial cells maybe collected from a human, bovine, or a mouse. Examples of the humansource for providing vascular endothelial cells include umbilical cordvein, umbilical cord artery, aorta, pulmonary artery, and blood vesselsof adult skin and neonatal foreskin. Culturing of such vascularendothelial cells is performed in a medium containing the aforementionedcancer cell culture supernatant. The volume of the cancer cell culturesupernatant to be added to the medium is preferably 10 to 100 v/v %,more preferably 20 to 100 v/v %. No particular limitation is imposed onthe medium in which vascular endothelial cells are cultured. Examples ofthe medium include, but are not limited to, RPMI 1640, MEM, DMEM, Ham'sF12, and M199, and any of these may be supplemented with, other than acancer cell culture supernatant, vascular endothelial cell growthfactor, heparin, an antibiotic, or a similar substance. No particularlimitation is imposed on the culture conditions under which vascularendothelial cells are cultured, but preferably, culturing is performedat 37° C., in an atmosphere of 5% CO₂ and 95% air, under saturated watervapor conditions for 2 to 4 days.

Examples of antigens derived from the aforementioned vascularendothelial cells include vascular endothelial cells per se,intracellular components of vascular endothelial cells (e.g., totalRNA), vascular endothelial cell lysates, and vascular endothelial cellmembrane vesicles. Intracellular components, cell lysates, and membranevesicles are preferred, as they are easy to prepare and effectivelystimulate dendritic cells.

A cell lysate may be prepared through, for example, low-speedcentrifugation (400 rpm for 10 minutes) of a cell product which has beenprepared by subjecting cells to 4 to 6 cycles of freezing (−160° C.) andthawing (37° C.). Also, a membrane vesicle may be prepared, for example,through the following process. Briefly, cells are treated overnight witha mixture containing DMEM and, as additives, 100 mM p-formaldehyde, 2 mMdithiothreitol, 1 mM CaCl₂, and 0.5 mM MgCl₂ at 37° C. in an atmosphereof 5% CO₂ and 95% air, under saturated water vapor conditions, followedby centrifugation for 5 minutes at 150×g. Afterwards, the supernatant iscentrifuged for 30 minutes at 30,000×g at 4° C. Intracellularcomponents, such as total RNA, may be prepared through a routine method.

The dendritic cells are preferably of human origin, and particularlypreferably are collected from the patient to whom the drug of thepresent invention is to be administered, or from a human who is MHCcompatible with the patient. Dendritic cells may be obtained from, forexample, myeloma cells, umbilical-cord-blood-derived cells, orperipheral mononuclear cells, through differentiation induced inaccordance with, for example, a method described in a non-patentdocument 8 or 9. In order to obtain dendritic cells from peripheralmononuclear cells by inducing differentiation, preferably, incubation isperformed in a medium supplemented with GM-CSF (10 to 100 ng/mL) andIL-4 (50 to 500 IU/mL) for 5 to 10 days.

Stimulation of dendritic cells with the above-described antigens derivedfrom vascular endothelial cells may be performed, for example, throughaddition of the vascular-endothelial-cell-derived antigens to asuspension of dendritic cells, and incubation of the mixture for 4 to 24hours. Preferably, cationic liposome or a similar substance is also usedin combination, to thereby improve the efficiency of stimulation withantigens. Furthermore, cell fusion of dendritic cells and vascularendothelial cells may be performed, so that vascular endothelial cellantigens are presented by the dendritic cells. Alternatively, total RNAof vascular endothelial cells may be introduced into dendritic cells, tothereby induce presentation of antigens. Preferably, the antigens areadded in an amount of 0.01 to 100 μg/mL, more preferably 0.1 to 100μg/mL, on a protein concentration basis per 1×10⁶ dendritic cells.

The thus-obtained dendritic cells are useful as a cancer remedy byvirtue of exerting the following functions. Briefly, they presentantigens specific for vascular endothelial cells of a cancer tissue inthe living organism, whereby cytotoxic T lymphocytes (CTLs) reactiveagainst vascular endothelial cells of the tumor tissue are induced.Thus, the vascular endothelial cells of the tumor tissue are attacked,and the blood vessels in the tumor tissue are destroyed. As a result,nutrition supply is cut, leading to retraction of the tumor.

In view that the cell suspension containing the above-prepared dendriticcells is administered to humans as a therapeutic drug for cancer, thesuspension is preferably subjected to a process for eliminating cellproliferative properties. For safer use of the drug, thedendritic-cell-containing suspension may be, for example, heated,radiated, or treated with mitomycin C, under mild conditions under whichonly cancer cell protein is denatured while the function of thedendritic cells as a therapeutic drug for cancer is retained. In thecase where X-ray irradiation is performed, for example, a flaskcontaining the dendric cells is placed under the tube of an X-rayradiation apparatus, and the flask is irradiated with X-rays at a totalradiation dose of 1,000 to 3,300 Rad. In the case where mitomycin Ctreatment is performed, for example, to a suspension containing thedendritic cells at a cell density of 1×10⁷ to 3×10⁷ cells/mL, mitomycinC is added in a ratio of 25 to 50 μg per mL of cell suspension, and theresultant mixture is left to stand at 37° C. for 30 to 60 minutes. Inthe case where heat treatment is performed, for example, a centrifugetube containing a cell suspension prepared to have a live cellconcentration of 1×10⁷ cells/mL is heated at 50 to 65° C. for 20minutes.

No particular limitation is imposed on the type of cancer to be treatedby the cancer therapeutic drug of the present invention. However, asolid cancer is preferred. Examples of the solid cancer include gastriccancer, liver cancer, colon cancer, lung cancer, skin cancer, bladdercancer, uterine cervical cancer, ovarian cancer, breast cancer, prostatecancer, and brain tumor. The amount of administration of the dendriticcells of the present invention varies depending on, for example,patient's age, body weight, and sex, as well as the identity and thestage of cancer, symptoms or the like, and cannot be determineduniformly. The dendritic cells of the present invention may beadministered to a patient in about the same amount as the injectionamount employed in the current cell vaccine therapy. The dendritic cellsof the present invention may be administered to a single intendedpatient, or alternatively, thanks to the well-established network ofbone marrow banks and umbilical cord blood banks, they may beadministered to numerous other patients having MHC compatibility.

EXAMPLES

The present invention will next be described in more detail by way ofExamples, which should not be construed as limiting the inventionthereto.

Example 1

A. Method

(1) Preparation of Cancer Cell Culture Supernatant (Cancer CM)

Mouse melanoma B16 cells were cultured in RPMI 1640 medium at 37° C. inan atmosphere of 5% CO₂ and 95% air under saturated water vaporconditions until subconfluency. The medium was replaced with a freshmedium (a culture medium for human umbilical cord vein endothelialcells, hereinafter referred to as HUVEC; composed of a mixture of Medium199 and RPMI 1640 in the ratio of 1:1 and supplemented with 15% fetalcalf serum, an endothelial cell proliferation factor (20 μg/mL), heparin(25 μg/mL), and an antibiotic). After 48 hours, the medium was recoveredand subjected to centrifugation at 1,000 rpm for 5 minutes. Thesupernatant was filtered by use of a 0.22 μm filter. The filtrate and afresh HUVEC medium were mixed in equal volumes, and the mixture was usedas a conditioned medium for cancer cells (cancer CM).

(2) Preparation of Cancer-CM-Reacted Vascular Endothelial Cells

The human umbilical cord vein endothelial cells were seeded in the HUVECmedium at a concentration of 2,500 cells/cm², and cultured at 37° C. inan atmosphere of 5% CO₂ and 95% air under saturated water vaporconditions. On the next day, the medium was replaced with the cancer CM.The umbilical cord vein endothelial cells were cultured in this mediumat 37° C. in an atmosphere of 5% CO₂ and 95% air under saturated watervapor conditions for 48 hours, and the cultured cells were used ascancer-CM-reacted vascular endothelial cells.

(3) Antigen for Pulsing Dendritic Cells

A membrane vesicle preparatory solution (DMEM in which 100 mMp-formaldehyde, 2 mM dithiothreitol, 1 mM CaCl₂, and 0.5 mM MgCl₂ weredissolved) was added to the cultured cancer-CM-reacted vascularendothelial cells for treatment of the cells overnight at 37° C.

The supernatant was then subjected to centrifugation at 150×g for 5minutes. Subsequently, the supernatant was subjected to centrifugationfor 30 minutes at 4° C. and 30,000×g. The resultant pellet was used asmembrane vesicles of the cancer-CM-reacted vascular endothelial cellsfor serving as an antigen for pulsing the dendritic cells.

Separately, cultured cancer-CM-reacted vascular endothelial cells weredissolved in saline to form a suspension, and the suspension wassubjected to four cycles of freezing and thawing. The cell lysate wasalso used as an antigen for pulsing the dendritic cells.

The membrane vesicles of the cancer-CM-reacted vascular endothelialcells or the cell lysate were mixed with lipofectin (on a volume basis)so as to attain a lipid concentration of 10 μg/mL, and the mixture wasleft to stand at room temperature for 20 minutes. The mixture was addedto a cell suspension (1×10⁶ cells/mL) of mouse dendritic cell strainDC2.4 cells or bone-marrow-derived, primary-cultured dendritic cells,and the cells were incubated for 5 hours. The resultant cells weresubjected to centrifugation using a phosphate buffer. After washing, thecells were treated with mitomycin C (50 μg/mL) at 37° C. for 30 minutes.Subsequently, the cells were subjected to centrifugation using thephosphate buffer twice, washed, and re-suspended in phosphate buffer.

(4) Administration of Dendritic Cells

The thus-prepared DC2.4 cells or the primary-cultured dendritic cellswere administered subcutaneously or intracutaneously in an amount of1×10⁵ cells to B57/BL6 mice.

(5) Administration of Cancer Cells

One week after the administration of the DC2.4 cells or theprimary-cultured dendritic cells, B16F10 cells (1×10⁶ cells) wereinjected into the tail vein.

(6) Evaluation of Lung Metastasis Incidence

One week after the administration of the B16F10 cells, the lung wasremoved, and the number of colonies which had spread to the lung wascounted under a stereoscopic microscope.

B. Results

The lung metastasis incidences of three mouse cases to which thedendritic cells had not been administrated were (924, 799, 550), and thelung metastasis incidences of three mouse cases to which the dendriticcells had been administrated were (184, 414, 0). The dendritic cellssignificantly suppressed the lung metastasis. The results have proventhat the dendritic cells of the present invention are useful as atherapeutic drug for cancer.

Example 2

A. Method

Bovine aorta endothelial cells (BAEC) which had been scraped off fromthe intima and subjected to subculture were employed. The mediumemployed for culturing the endothelial cells is DMEM supplemented with10% fetal calf serum. The endothelial cells were processed in the samemanner as in Example 1, to thereby produce cell lysate serving as anantigen.

Through the same procedure as in Example 1, except that a mediumcontaining a supernatant of B16 cell culture was used, thebovine-aorta-derived endothelial cells were cultured. The dendriticcells were pulsed with the cell lysate of the cultured cells, whichserved as an antigen. The pulsed dendritic cells were administered toB57/BL6 mice by the same method as in Example 1. Two weeks after thecancer cell administration, the lung was removed, and the lung weightwas measured.

B. Results

As shown in FIG. 1, in the cases of the “None” group (administration ofB16 only), the “None pulsed DC” group (administration of B16 and thenon-pulsed dendritic cells), and the “BAEC pulsed DC” group(administration of B16 and the BAEC pulsed dendritic cells), almost allportions of the lung were found to be black due to B16, and the lungweight was also found to be increased. In contrast to the above, in thecase of the “B16CM-BAEC pulsed DC” group (administration of B16 and thedendritic cells which had been pulsed with BAEC cultured in B16 culturesupernatant), the black areas in the lung were found to be very limited,and an increase in the lung weight was also found to be suppressed.

1. A therapeutic drug for cancer, comprising, as an active ingredient,dendritic cells stimulated with an antigen derived from vascularendothelial cells which have been cultured in a medium containing acancer cell culture supernatant.
 2. The therapeutic drug for canceraccording to claim 1, wherein the antigen derived from vascularendothelial cells comprises a vascular endothelial cell, anintracellular component of a vascular endothelial cell, a vascularendothelial cell lysate, or a vascular endothelial cell membranevesicle.
 3. Use, in production of a therapeutic drug for cancer, ofdendritic cells stimulated with an antigen derived from vascularendothelial cells which have been cultured in a medium containing acancer cell culture supernatant.
 4. The use according to claim 3,wherein the antigen derived from vascular endothelial cells comprises avascular endothelial cell, an intracellular component of a vascularendothelial cell, a vascular endothelial cell lysate, or a vascularendothelial cell membrane vesicle.
 5. A therapeutic method for cancer,which comprises administering dendritic cells stimulated with an antigenderived from vascular endothelial cells which have been cultured in amedium containing a cancer cell culture supernatant.
 6. The therapeuticmethod according to claim 5, wherein the antigen derived from vascularendothelial cells comprises a vascular endothelial cell, anintracellular component of a vascular endothelial cell, a vascularendothelial cell lysate, or a vascular endothelial cell membranevesicle.